Microorganism sampling method, microorganism sampling device and sampling kit comprising such a sampling device

ABSTRACT

A sampling method using a sampling device ( 2 ) comprising a container ( 5 ) comprising a body ( 6 ) consisting of a soft pouch, and a lid ( 30 ), in which at least one among the container ( 5 ) and the lid ( 30 ) is provided with a discharging member ( 40 ) suitable for, in an open state, discharging at least one part of the gases contained in the internal space ( 7 ) of the body ( 6 ) of the container ( 5 ), wherein the sampling method comprises the steps consisting in:—collecting a biological material in the body ( 6 ) of the container ( 5 ), and assembling the lid ( 30 ) on the neck ( 10 ) of the container ( 5 ),—and placing an internal space ( 7 ) of the body ( 6 ) of the container ( 5 ) under anaerobic conditions by placing the discharging member ( 40 ) in the open state, by compressing the body ( 6 ) of the container ( 5 ) and by placing the discharging member ( 40 ) in a closed state.

The invention relates to a microorganism sampling method, amicroorganism sampling device and a sampling kit employing such asampling device

The invention applies in particular to the sampling of human intestinalmicrobiota for example for testing intestinal dysbioses, such asinfections of Clostridium difficile, by transportation of the intestinalmicrobiota.

The intestinal microbiota is constituted by the group of allmicroorganisms (bacteria, yeast and fungi) to be found in the humangastro-intestinal tract (intestine, stomach and stools). The microbialdiversity is currently estimated at about 10³ bacterial speciescomposing the dominant intestinal microbiota of an adult individual,with an abundance of 10¹⁴ bacteria, representing a bacterial metagenomeof 200 000 to 800 000 genes in each individual, which is 10 to 50 timesthe number of genes of the human genome.

The intestines are sterile in utero and are colonized as of the firstdays of life to develop towards a unique individual microbiota. Eachperson has bacteria that are relatively close in terms of species, butthe exact composition of his or her microbiota (species, proportions) isto a large extent (about ⅔ of the species) specific to the host.

Thus, the human intestinal microbiota is a very diversified ecosystem,which is complex and specific to each individual.

It is essential for the health of an individual to maintain a stablemicrobiota which is both capable of returning to its initial state aftera change and resistant to invasion. Maintaining a wide diversity ofmicrobiota promotes its stability.

However, certain pathologies or treatments unbalance the microbiota: forexample, antibiotics as well as diseases with an inflammatory component,such as inflammatory bowel disease (IBD), can limit the diversity of themicrobiota in the intestines. Antibiotics treatments (or antibiotictherapy), in particular, result in an alteration of the microbiota,which can promote the proliferation of pathogenic organisms such asClostridium difficile. Infections of Clostridium difficile areresponsible for nosocomial diarrhea; this bacterium is resistant toconventional antibiotic therapy (of broad spectrum, such as vancomycinand metronidazole).

In order to reestablish the intestinal flora, and fight againstinfections of Clostridium difficile type, and thereby reestablishhomeostasis (i.e. symbiosis), a transplantation of faecal microbiota hasbeen envisioned and tested. It consists in the introduction of thestools of a healthy donor subject into the digestive tract of arecipient patient, in order to re-balance the altered intestinal floraof the host. This transplantation of faecal microbiota can be allogenic(that is to say from a healthy donor individual to a patient) orautologous (that is to say from an individual to himself). The resultsobtained on infections of Clostridium difficile type are encouraging,and some patients have been successfully treated (Tauxe et al, LabMedicine, Winter 2015, volume 46, Number 1).

Prior to performing such a transplantation, the microbiota is generallysampled from the healthy donor subject through use of a sampling deviceof the type comprising:

-   -   a container comprising a body which comprises an internal space        configured to receive the biological matter, and a neck which        delimits an access opening to the internal space of the body,        the body of the container being constituted by a flexible bag,    -   a cover configured to be removably and sealingly mounted on the        neck of the container so as to obturate the access opening of        the neck and close the internal space of the body,    -   a transfer member internally comprising a passage between the        internal space and an external environment, the transfer member        having an open state in which said transfer member establishes        communication with the internal space through the passage, and a        closed state in which said transfer member prevents all        communication with the internal space through the passage.

A known sampling device of this type is described in document WO2013/090825. With such a sampling device, after having collected thebiological matter, the container is closed with the cover before placingthe biological matter in suspension by mixing the collected biologicalmatter with a diluent fluid. Anaerobic conditions may be obtained in theinternal space of the body by suction, possibly complemented by theaddition of appropriate substances.

However, the known sampling device does not provide the conditionsenabling a simple way of providing satisfactory preservation of thebacteria of the microbiota, and especially of the anaerobic bacteria,which are the majority components of the intestinal microbiota.Furthermore, the known sampling device does not make it possible toperform the necessary manipulations for sampling the microbiota in asafe way, the operator performing the manipulations running the risk ofentering into contact with the biological matter.

The invention is directed to mitigating the problems raised above.

To that end, according to a first aspect, the invention provides asampling method for sampling microorganisms contained in biologicalmatter, the sampling method employing a sampling device of theaforementioned type wherein said at least one transfer member comprisesan evacuation member configured for evacuating, in the open state, atleast part of the gases contained in the internal space of the body ofthe container, the sampling method comprising the steps consisting of:

-   -   collecting the biological matter in the internal space of the        body of the container, and closing the internal space by        mounting the cover on the neck of the container,    -   putting the internal space of the body of the container under        anaerobiosis by putting the evacuation member in the open state,        by compressing the body of the container so as to evacuate at        least part of the gases contained in the internal space of the        body of the container by putting the evacuation member in the        closed state.

Thus, the invention enables optimal conditions for the preservation ofmicroorganisms, and in particular the microbiota, to be obtained simplyand safely. In particular, the invention enables placing underanaerobiosis to be performed by simple compression applied on the bodyof the container to evacuate the oxygen and preserve the anaerobicbacteria that are sensitive to oxygen. The bacteria maintained in aclosed system may be kept in these conditions of anaerobiosis whilebeing isolated from external contaminants. Furthermore, this placingunder anaerobiosis as well as the later placing in suspension of thebiological matter that are carried out by compressing the body of thecontainer, without having recourse to a mixer, enable the viability ofthe bacteria to be enhanced. Furthermore, the manipulations may becarried out with the container closed by the cover and thus without riskof direct contact with the biological matter for the operator.

the evacuation member may have opposite internal and external faces, theevacuation member being in the closed state when at rest and passinginto the open state when a difference of pressure is applied between theinternal and external faces. the sampling method may then provide,during the step consisting of putting the internal space of the body ofthe container under anaerobiosis, of automatically putting theevacuation member in the open state by compressing the body of thecontainer then automatically putting the evacuation member in the closedstate by stopping compressing the body of the container.

the cover may comprise a bearing part having overall rigidity and theevacuation member may be provided in the bearing part of the cover. Thesampling method may then provide, during the step consisting of puttingthe internal space of the body of the container under anaerobiosis, forpressing the body of the container against the bearing part of thecover.

The sampling method may further comprise the step consisting ofsuspending the biological matter by introducing a diluent fluid into theinternal space of the body of the container via said at least onetransfer member in the open state, and by mixing the biological matterand the diluent fluid by pressing on the body of the container.

The sampling method may further comprise the step consisting of samplingthe microorganisms by collecting at least some of the biological mattersuspended via said at least one transfer member in the open state.

The sampling method may provide, during the step consisting ofcollecting the biological matter in the internal space of the body ofthe container, for directly collecting the faecal matter by placing thesampling device on a toilet seat.

According to a second aspect, the invention provides a sampling devicefor sampling microorganisms contained in biological matter, the samplingdevice comprising:

-   -   a container comprising a body which comprises an internal space        configured to receive the biological matter, and a neck which        delimits an access opening to the internal space of the body,        the body of the container being constituted by a flexible bag,    -   a cover configured to be removably and sealingly mounted on the        neck of the container so as to obturate the access opening of        the neck and close the internal space of the body,

wherein at least one of the container and the cover is provided with atleast one transfer member internally comprising a passage between theinternal space and an external environment, the transfer member havingan open state in which said transfer member establishes communicationwith the internal space through the passage, and a closed state in whichsaid transfer member prevents all communication with the internal spacethrough the passage,

wherein said at least one transfer member comprises an evacuation memberhaving opposite internal and external faces, the evacuation member beingin the closed state when at rest and passing into the open state when adifference of pressure is applied between the internal and externalfaces, so as to automatically put the evacuation member in the openstate by compressing the body of the container to evacuate at least partof the gases contained in the internal space of the body of thecontainer and place the internal space of the body of the containerunder anaerobiosis, and to automatically put the evacuation member inthe closed state by stopping compressing the body of the container.

To avoid spreading germs and thus ensure the safety of the manipulator,the evacuation member may comprise a microporous retaining membranedisposed in the passage and configured to retain the biological matterwhile allowing gases to pass

Said at least one transfer member may comprise a connection portprovided through one of the container and the cover and internallycomprising at least part of the passage, and an obturating membermovable relative to the connection port between an obturating position,in which said obturating member prevents all communication through thepassage, and a freeing position in which said obturating member allowscommunication through the passage.

The obturating member may be mounted in the passage of the connectionport, the obturating member being urged towards its obturating positionand being movable towards the freeing position These provisions make itpossible to make a connection simply and without boring, which isremovable and fluid-tight and which, where required, enablesbidirectional communication while maintaining the internal spaceentirely closed so long as the obturating member is not moved by apositive action towards the freeing position.

Said at least one transfer member may comprise at least one tubeextending from the connection port and internally comprising part of thepassage.

Said at least one transfer member may comprise at least one valvemounted on the tube, the valve being movable between a closed position,in which said valve prevents all communication through the passage, andan open position in which said valve allows the communication throughthe passage.

When the obturating member is mounted in the passage of the connectionport, the tube may comprise a complementary connection port providedwith a freeing member and which is configured to be removably connectedto the connection port such that the freeing member moves the obturatingmember towards the freeing position.

The cover may comprise a bearing part having overall rigidity and theevacuation member may be provided in the bearing part of the cover, soas to be able to press the body of the container against the bearingpart of the cover to put the internal space of the body of the containerunder anaerobiosis.

The sampling device may further comprise a filter attached to thecontainer so as to define, in the internal space, an upper compartmentinto which the access opening opens, and a lower compartment, the filterhaving pores comprised between 0.1 mm and 1.5 mm.

In order to avoid obstruction of the passage by a solid body containedin the biological matter or sampling of such a solid body with themicroorganisms, said at least one transfer member may be provided in thelower compartment, downstream of the filter relative to the accessopening.

According to a third aspect, the invention concerns a sampling kitcomprising:

-   -   a sampling device as defined above, and    -   at least one ancillary device chosen from a supply device        configured to supply the internal space with fluid, and a        receiving device configured to receive a fluid contained in the        internal space, the ancillary device being in particular chosen        from a reservoir of diluent fluid, an analysis tube, a        distribution pipe and a collecting bag for microorganisms.

The sampling kit makes it possible in particular to sample and processthe microorganisms into the form of an inoculum ready for use.

When the transfer member of the sampling device comprises an obturatingmember mounted in the passage of a connection port, the ancillary devicemay comprise a complementary connection port provided with a freeingmember and which is configured to be removably connected to theconnection port such that the freeing member moves the obturating membertowards the freeing position.

Other objects and advantages of the invention will appear on reading thefollowing description of a specific embodiment of the invention given byway of non-limiting example, the description being made with referenceto the accompanying drawings in which:

FIG. 1 is a representation in perspective of a sampling kit comprising asampling device for sampling microbiota contained in a biologicalmatter, the sampling device comprising a container and a cover, thecontainer comprising a body constituted by a flexible bag and the coverbeing provided with an evacuation member configured to evacuate at leastpart of the gases contained in an internal space of the body of thecontainer,

FIG. 2 is a representation in perspective of the sampling device of FIG.1 in an application for sampling intestinal microbiota contained infaecal matter such as biological matter, the sampling device being openwith the cover separate from the container for collecting the faecalmatter,

FIG. 3 is a representation in perspective of the sampling device of FIG.1 placed in a condition of anaerobiosis with the cover mounted on thecontainer and the body pressed against the cover to evacuate the oxygenfrom the internal space of the body and preserve the microbiota presentin the collected faecal matter,

FIG. 4 is a flowchart illustrating steps of a method of samplingmicrobiota contained in biological matter, the sampling methodimplementing the sampling device of FIG. 1.

In the drawings, the same references designate identical or similarparts.

FIG. 1 represents an embodiment of a sampling kit 1 comprising asampling device 2 for sampling microorganisms contained in biologicalmatter. In the embodiment represented, without being limited thereto,the sampling device 2 is implemented in the sampling of intestinalmicrobiota contained in the faecal matter of an individual.

The sampling device 2 comprises a container 5 adapted in particular tocollect faecal matter.

The container 5 comprises a body 6 constituted by a flexible bagdelimiting an internal space 7 configured to receive the biologicalmatter and having an upper edge 8. The body 6 is formed from adeformable material configured to form a barrier to the faecal mattercontained in the internal space 7 and to external fluids in order toavoid any exchange between the faecal matter and the exterior. Inparticular, the body 6 may be produced from polyethylene (PE),polypropylene (PP), polyvinyl chloride (PVC), polycarbonate (PC) orethylene-vinyl acetate (EVA).

The container 5 also comprises a cylindrical neck 10, defined by a solidof revolution about a central axis A, to which the upper edge 8 of thebody 6 is joined by any appropriate means and in particular by welding,bonding or other means. The neck 10 has overall rigidity and is, forexample, produced from plastic material such as polyethylene (PE),polypropylene (PP), polyvinyl chloride (PVC), polycarbonate (PC) orethylene-vinyl acetate (EVA). The neck 10 has an internal surface 11which delimits an access opening 12 to the internal space 7 of the body6, and an external surface 13 provided with one or more ramps 14arranged so as to form an external screw thread. As a variant, any othercontinuous or discrete arrangement of one or more ramps 14, which may beequally spaced, could be provided on the external surface 13 of the neck10.

The container 5 also comprises a support 15 adapted to provide stableholding in an appropriate orientation, in particular with the accessopening 12 upwardly oriented. In the embodiment represented, the support15 is joined to the neck 10 and shaped so as to be able to come to bearon a rim of a toilet seat 3 in accordance with the applicationconsidered. The support 15 comprises two wings 16 extending radiallyrelative to the central axis A in diametrically opposite directions fromthe external surface 13 of the neck 10. As a variant, according to theapplication, the support could have any other appropriate shape.

A filter 20 is joined, for example by welding, bonding or other method,to the container 5 so as to define, in the internal space 7, twocompartments. In FIG. 1, the filter 20 is then joined to the body 6 soas to extend in the neighborhood of the access opening 12 of the neck10. The internal space 7 then has an upper compartment into which opensthe access opening 12 and a lower compartment in the region of thebottom 9. As a variant, the filter may be joined at any otherappropriate location of the neck or of the body of the container todefine lower and upper compartments of appropriate respectivecapacities. The filter has pores comprised between 0.1 mm and 1.5 mm, inparticular between 0.3 mm and 0.5 mm, configured to retain insolublesolid bodies contained in the faecal matter.

The body 6 of the container 5 comprises a first transfer member forming,in the particular embodiment represented, a collecting member 25enabling the microorganisms from the intestinal microbiota to becollected. The collecting member 25 comprises a lower connection port 26formed through the body 6 of the container 5 in the neighborhood of thebottom 9 of the body 6, and a tube 27 extending from the lowerconnection port 26. The lower connection port 26 and the tube 27 therebydefine a passage inside the collecting member 25 between the internalspace 7 and an external environment. The collecting member 25 comprisesa valve 28 mounted on the tube 27 and movable between:

-   -   a closed position defining a closed state of the collecting        member 25 in which it prevents any communication, and in        particular any fluid flow, through the passage, and    -   an open position defining an open state of the collecting member        25 in which it allows communication, and in particular fluid        flow, through the passage.

As a variant, the collecting member 25 could comprise several tubes 27and several valves 28.

The tube 27 may be removably linked to the lower connection port 26. Anobturating member in the form of a check valve may be mounted in thepassage of the lower connection port 26 and be urged towards anobturating position, in which the obturating member prevents anycommunication through the passage, and be movable towards a freeingposition in which the obturating member allows the communication throughthe passage. The tube 27 then comprises a complementary connection portconfigured to cooperate with the lower connection port 26. Inparticular, the complementary connection port is provided with a freeingmember arranged to move the obturating member to the freeing positionwhen the complementary connection port is connected to the lowerconnection port 26. The lower connection port 26 and the complementaryconnection port may then form, one being the male member and the otherthe female member, a Luer Lock type connection equipped with a checkvalve and in which:

-   -   the lower connection port 26 is in the obturating position        preventing any communication between the internal space 7 and        the exterior through the passage when the tube 27 is separated        from the body 6 of the container 5, and    -   the lower connection port 26 is in the freeing position        establishing communication between the internal space 7 and the        exterior through the passage when the tube 27 is connected to        the body 6 of the container 5.

Such a connection port makes it possible, by a connection withoutpiercing, to form a two-directional communication through the passagewhile maintaining the internal space totally closed so long as theobturating member has not been moved towards the freeing position by thefreeing member.

In another embodiment, the tube 27 of the collecting member 25 may beintegrally formed with the lower connection port 26, the valve 28 thenforming the obturating member movable relative to the connection port 26between an obturating position corresponding to the closed position ofthe valve 28, and a freeing position corresponding to the open position.As a variant, instead of the valve 28, the obturating member could be aplug that can be pierced or that is removably mounted on a free end ofthe tube 27.

In order to be able to preserve the collected faecal matter, thesampling device 2 comprises a cover 30 adapted to be mounted removablyand in fluid-tight manner on the neck 10 of the container 5 so as toobturate the access opening 12 of the neck 10 and to close the internalspace 7 of the body 6. The cover 30 has overall rigidity and is, forexample, formed from a plastic material similar to that of the neck 10.It comprises a transverse wall 31 extending perpendicularly to thecentral axis B and having a circular peripheral edge 32 with a diametercorresponding to that of the neck 10 of the container 5. The cover 30also comprises a cylindrical skirt 33, defined as a solid of revolutionabout the central axis B, which extends from the peripheral edge 32 ofthe transverse wall 31. The skirt 33 has an internal surface providedwith projections which, according to requirement, form an internal screwthread, and which are configured to cooperate with the ramps 14 on theexternal surface 13 of the neck 10. As a variant, any other deviceenabling the cover 30 and the neck 10 of the container 5 to be joinedremovably and in a fluid-tight manner could be provided, in particular aclipping device. The transverse wall 31 has an upper surface having tworecesses 34 arranged on respective opposite sides of a centralprojection 35 for grasping. The recesses 34 are such that a lowersurface of the transverse wall 31 at the location of the recesses 34 isflush with or extends beyond a free edge 36 of the skirt 33.

In one of the recesses 34, the cover 30 is provided with a secondtransfer member produced in the form of an evacuation member configured,in an open state, to evacuate at least part of the gases contained inthe internal space 7 of the body 6 of the container 5. In the particularembodiment represented, the evacuation member is a one-way valve 40which is in a closed state at rest, when not acted upon externally, andwhich passes to an open state when a difference of pressure is appliedbetween opposite internal and external faces, respectively situated atthe internal and external surfaces of the cover 30.

In particular, the one-way valve 40 comprises an upper connection port41, formed through the cover 30, between its lower and upper surfaces,and a tube 42 extending from the upper connection port 41. The upperconnection port 41 and the tube 42 thereby define a passage inside theone-way valve 40 between the internal space 7 and the externalenvironment. In a central bead 43, the tube 42 of the one-way valve 40comprises a microporous retaining membrane disposed in the passage andwhich is configured to retain the biological matter while enabling gasesto pass. The microporous retaining membrane may in particular be of thetype commercialized by POREX® FILTRATION under the reference XS-49 110made of polypropylene with pores comprised between 125 μm and 175 μm. Asa variant, the microporous retaining membrane could be produced in anyother appropriate way and in particular of a sintered material havingpores comprised between 100 μm and 350 μm, in particular between 200 μmand 300 μm, for example 250 μm.

An obturating member in the form of a check valve may be mounted in thepassage of the one-way valve 40 and be urged towards an obturatingposition, preventing any communication through the passage, and bemovable towards a freeing position allowing communication through thepassage.

In the embodiment shown, the tube 42 of the one-way valve 40 isintegrally formed with the upper connection port 41. The obturatingmember may be provided in the tube 42, in particular in the neighborhoodof a free end.

In another embodiment, the tube 42 of the one-way valve 40 may beremovably connected to the upper connection port 41. In addition to orinstead of the check valve provided in the tube 42, a new obturatingmember in the form of a check valve may be provided in the upperconnection port 41 and be urged towards an obturating position,preventing any communication through the passage, and be movable towardsa freeing position allowing communication through the passage. In thethis other embodiment, the tube 42 comprises a complementary connectionport adapted to cooperate with the upper connection port 41. Inparticular, the complementary connection port is provided with a freeingmember arranged to move the check valve of the upper connection port 41to the freeing position when the complementary connection port isconnected to the upper connection port 41. The upper connection port 41is in the obturating position when the tube 42 is separated from thecover 30 and in the freeing position when the tube 42 is connected tothe cover 30.

As a variant, the evacuation member could be formed in any appropriateway other than a one-way valve. In particular, the evacuation membercould comprise a passage able to be obturated by an obturating member inthe form of a plug removably placed in the passage, for example by beingremovably mounted on the free end of the tube 42. When the plug isremoved from the passage, the evacuation member is in the open state, inwhich communication between the internal space 7 of the container 5 andthe exterior is established through the passage. When the plug is placedin the passage, it obturates the passage and the evacuation member is ina closed state, in which any communication between the internal space 7of the container 5 and the exterior through the passage is prevented.

In the other recess 34, the cover 30 comprises third and fourth transfermembers which respectively form, in the particular embodiment shown, asupply member 45 and a sampling member 48. The supply member 45 andsampling member 48 are each adapted to establish communication betweenthe internal space and the exterior in the open state, and to close thecommunication between the internal space and the exterior in the closedstate.

The supply members 45 and sampling member 48 respectively compriseadditional upper connection ports 46, 49 for ancillary devices of thesampling kit.

Each of the additional upper connection ports 46, 49 is formed throughthe cover 30, between the lower and upper surfaces, to constitute one ofthe members, for example female, of a Luer Lock type connection. As wasdescribed above, each of the additional upper connection ports 46, 49 ofthe supply member 45 and sampling member 48 may be provided with anobturating member produced in the form of a check valve 47, a plug orother member.

The obturating member of the supply member 45 is, for example, producedin the form of a check valve 47 mounted in the passage and configured tocooperate with a freeing member of a complementary connection portprovided on the ancillary device and constituting the other member, forexample male, of the Luer Lock type connection. The check valve 47 urgedtowards an obturating position preventing any communication through thepassage places the additional upper connection port 46 of the supplymember 45 in a closed state when the ancillary device is separated fromthe cover 30. The obturating member 47 moved to a freeing positionpermitting communication through the passage places the additional upperconnection port 46 of the supply member 45 in an open state when theancillary device is connected to the cover 30.

The obturating member of the sampling member 48 is, for example,produced in the form of a plug, not shown, removably mounted on theadditional upper connection port 49. The sampling member 48 is in anopen state promoting communication through the passage when the plug isseparated from the additional upper connection port 49, and a closedstate preventing any communication through the passage when the plug ison the additional upper connection port 49.

The ancillary devices may in particular comprise:

-   -   one or more reservoirs of diluent fluid, and in particular a        supply bag, configured to be connected to the additional upper        connection port 46 of the supply member 45, the reservoir        comprising, where required, a pipe equipped with a complementary        connection port with a freeing member.    -   one or more analysis tubes configured to be connected to the        additional upper connection port 49 of the sampling member 48,        each analysis tube extending between an open end provided, where        required, with a complementary connection port with a freeing        member, and a closed end so as to be able to extract faecal        matter for analysis purposes, and    -   one or more bags for collecting microorganisms configured to be        connected to the lower connection port 26 of the collecting        member 25 via a distribution duct comprising one or more pipes        equipped with a complementary connection port with a freeing        member and, where required, with one or more valves.

As a variant, the sampling kit 1 could comprise any other type ofancillary device chosen from a supply device configured to supply theinternal space 7 with fluid, and a receiving device configured toreceive a fluid contained in the internal space 7.

The invention has been described with a sampling device comprisingfirst, second, third and fourth transfer members respectivelyconstituting a collecting member, an evacuation member in the form of aone-way valve, a supply member and a sampling member. As a variant, itwould be possible to provide any other arrangement, any other embodimentand any other functionality with one or more transfer members internallycomprising a passage and of which at least one forms an evacuationmember configured, in the open state, to evacuate at least part of thegases contained in the internal space of the body of the container. Inparticular, the sampling device 2 could comprise only the first 25 andsecond 40 transfer members described earlier or for instance only one ofthem. At least one transfer member is provided, preferably in the lowercompartment, downstream of the filter relative to the access opening, inorder to avoid obstruction of the connection port by a solid bodycontained in the biological matter or a sample of such a solid body withthe microorganisms.

A method of sampling microbiota contained in biological matterimplementing the sampling system will now be described in relation withFIGS. 2 to 4.

In FIG. 2, faecal matter is collected by placing the container 5 of thesampling device 2 on the toilet seat 3 with the wings resting on the rimof the toilet seat 3. As a variant, the faecal matter could be depositedin any other appropriate way in the internal space 7 of the container 5.During this collecting step, the tube 27 may be separated from thecontainer 5.

In FIG. 3, once the faecal matter has been received on the filter 20 inthe internal space 7 of the body 6, the internal space 7 is closed byscrewing the cover 30 onto the neck 10 of the container 5. Thecollecting member 25, the one-way valve 40 and the two sampling members45, 48 are in the closed state. An operator may then proceed withplacing the internal space 7 of the body 6 of the container 5 underanaerobiosis by compressing the body 6 of the container 5 therebyautomatically passing the one-way valve 40 into the open state. Inparticular, the transverse wall 31 of the cover 30 at the location ofthe recesses 34 forms a bearing part against which the body 6 of thecontainer 5 can be pressed when the cover 30 is mounted on the neck 10of the container 5. In doing this, part of the gases contained in theinternal space 7 of the body 6 of the container 5, and in particular theoxygen, is evacuated through the one-way valve 40 provided in thebearing part of the cover 30. The compression of the body 6 may beimproved by a grip or rest on a gripping member, such as a rigid plateor a handle, arranged on a base 9 at the remote opposite to the upperedge 8. Once the compression of the body 6 of the container 5 isstopped, the one-way valve 40 automatically passes back to the closedstate to maintain the anaerobic conditions in the internal space 7 ofthe body 6.

According to the application considered, the placing under anaerobiosismay be completed by an injection of inert gas into the internal space 7of the body 6 of the container 5 via one of the transfer members 25, 40,45, 48.

After a possible waiting time under determined conditions and possiblevisual inspection, the faecal matter is placed in suspension byconnecting the reservoir of diluent fluid, such as a bag of diluentfluid or a bottle of diluent fluid, to the additional upper connectionport 46 of the supply member 45 on the cover 30 of the sampling device2. The additional upper connection port 46 passes from the open statesuch that the diluent fluid is introduced into the internal space 7 ofthe body 6 of the container 5. The biological matter and the diluentfluid are then mixed by pressing on the body 6 of the container 5. Thehomogeneity of the suspension is improved by its passage through thefilter 20.

Prior to the placing in suspension of the faecal matter, complementaryanalyses may be performed on a sample taken via an analysis tubeconnected to the additional upper connection port 49 of the samplingmember 48 on the cover 30 of the sampling device 2. As a variant, thesampling of such a sample may be performed simultaneously orsubsequently to the placing in suspension.

To process the microbiota into the form of an inoculum ready for use,the tube 27 or a distribution duct may be connected to the lowerconnection port 26 of the collecting member 25 to take off themicrobiota contained in the faecal matter to one or more collecting bagsrespectively connected to one or more pipes of the distribution duct.

The microbiota may then undergo any appropriate operation for itspreservation, possible transport and its transplantation.

1. A sampling method for sampling microorganisms contained in biologicalmatter, the sampling method employing a sampling device (2) comprising:a container (5) comprising a body (6) which comprises an internal space(7) configured to receive the biological matter, and a neck (10) whichdelimits an access opening (12) to the internal space (7) of the body(6), the body (6) of the container (5) being constituted by a flexiblebag, a cover (30) configured to be removably and sealingly mounted onthe neck (10) of the container (5) so as to obturate the access opening(12) of the neck (10) and close the internal space (7) of the body (6),wherein at least one of the container (5) and the cover (30) is providedwith at least one transfer member (25, 40, 45, 48) internally comprisinga passage between the internal space (7) and an external environment,the transfer member (25, 40, 45, 48) having an open state in which saidtransfer member (25, 40, 45, 48) establishes communication with theinternal space (7) through the passage, and a closed state in which saidtransfer member (25, 40, 45, 48) prevents all communication with theinternal space (7) through the passage, said at least one transfermember (25, 40, 45, 48) comprising an evacuation member (40) configuredfor evacuating, in the open state, at least part of the gases containedin the internal space (7) of the body (6) of the container (5), thesampling method comprising the steps consisting of: collecting thebiological matter in the internal space (7) of the body (6) of thecontainer (5), and closing the internal space (7) by mounting the cover(30) on the neck (10) of the container (5), putting the internal space(7) of the body (6) of the container (5) under anaerobiosis by puttingthe evacuation member (40) in the open state, by compressing the body(6) of the container (5) so as to evacuate at least part of the gasescontained in the internal space (7) of the body (6) of the container (5)by putting the evacuation member (40) in the closed state.
 2. A samplingmethod according to claim 1, wherein the evacuation member (40) hasopposite internal and external faces, the evacuation member being in theclosed state when at rest and passing into the open state when adifference of pressure is applied between the internal and externalfaces, the sampling method providing, during the step consisting ofputting the internal space (7) of the body (6) of the container (5)under anaerobiosis, of automatically putting the evacuation member (40)in the open state by compressing the body (6) of the container (5) thenautomatically putting the evacuation member (40) in the closed state bystopping compressing the body (6) of the container (5).
 3. A samplingmethod (2) according to claim 1, wherein the cover (30) comprises abearing part (31) having overall rigidity and wherein the evacuationmember (40) is provided in the bearing part (31) of the cover (30), thesampling method providing, during the step consisting of putting theinternal space (7) of the body (6) of the container (5) underanaerobiosis, for pressing the body (6) of the container (5) against thebearing part (31) of the cover (30).
 4. A sampling method according toclaim 1, further comprising the step consisting of suspending thebiological matter by introducing a diluent fluid into the internal space(7) of the body (6) of the container (5) via said at least one transfermember (25, 40, 45, 48) in the open state, and by mixing the biologicalmatter and the diluent fluid by pressing on the body (6) of thecontainer (5).
 5. A sampling method according to claim 4, furthercomprising the step consisting of sampling the microorganisms bycollecting at least some of the biological matter suspended via said atleast one transfer member (25, 40, 45, 48) in the open state.
 6. Asampling method according to claim 1, providing, during the stepconsisting of collecting the biological matter in the internal space (7)of the body (6) of the container (5), for directly collecting the faecalmatter by placing the sampling device on a toilet seat (3).
 7. Asampling device (2) for sampling microorganisms contained in biologicalmatter, the sampling device (2) comprising: a container (5) comprising abody (6) which comprises an internal space (7) configured to receive thebiological matter, and a neck (10) which delimits an access opening (12)to the internal space (7) of the body (6), the body (6) of the container(5) being constituted by a flexible bag, a cover (30) configured to beremovably and sealingly mounted on the neck (10) of the container (5) soas to obturate the access opening (12) of the neck (10) and close theinternal space (7) of the body (6), wherein at least one of thecontainer (5) and the cover (30) is provided with at least one transfermember (25, 40, 45, 48) internally comprising a passage between theinternal space (7) and an external environment, the transfer member (25,40, 45, 48) having an open state in which said transfer member (25, 40,45, 48) establishes communication with the internal space (7) throughthe passage, and a closed state in which said transfer member (25, 40,45, 48) prevents all communication with the internal space (7) throughthe passage, the sampling device (2) being characterized in that said atleast one transfer member (25, 40, 45, 48) comprises an evacuationmember (40) having opposite internal and external faces, the evacuationmember being in the closed state when at rest and passing into the openstate when a difference of pressure is applied between the internal andexternal faces, so as to automatically put the evacuation member (40) inthe open state by compressing the body (6) of the container (5) toevacuate at least part of the gases contained in the internal space (7)of the body (6) of the container (5) and place the internal space (7) ofthe body (6) of the container (5) under anaerobiosis, and toautomatically put the evacuation member (40) in the closed state bystopping compressing the body (6) of the container (5).
 8. A samplingdevice (2) according to claim 7, wherein the cover (30) comprises abearing part (31) having overall rigidity and wherein the evacuationmember (40) is provided in the bearing part (31) of the cover (30), soas to be able to press the body (6) of the container (5) against thebearing part (31) of the cover (30) to put the internal space (7) of thebody (6) of the container (5) under anaerobiosis.
 9. A sampling device(2) according to claim 7, wherein the evacuation member (40) comprises amicroporous retaining membrane disposed in the passage and configured toretain the biological matter while allowing gases to pass.
 10. Asampling device (2) according to claim 7, wherein said at least onetransfer member (25, 40, 45, 48) comprises a connection port (26, 41,46, 49) provided through one of the container (5) and the cover (30) andinternally comprising at least part of the passage, and an obturatingmember (47) movable relative to the connection port (26, 41, 46, 49)between an obturating position, in which said obturating member (47)prevents all communication through the passage, and a freeing positionin which said obturating member (47) allows communication through thepassage.
 11. A sampling device (2) according to claim 10, wherein theobturating member (47) is mounted in the passage of the connection port(26, 46), the obturating member (47) being urged towards its obturatingposition and being movable towards the freeing position.
 12. A samplingdevice (2) according to claim 10, wherein said at least one transfermember (25, 40) comprises at least one tube (27, 42) extending from theconnection port (26, 41) and internally comprising part of the passage.13. A sampling device (2) according to claim 12, wherein said at leastone transfer member (25) comprises at least one valve (28) mounted onthe tube (27), the valve (28) being movable between a closed position,in which said valve (28) prevents all communication through the passage,and an open position in which said valve (28) allows the communicationthrough the passage.
 14. A sampling device (2) according to claim 18,wherein the tube (27) comprises a complementary connection port providedwith a freeing member and which is configured to be removably connectedto the connection port (26) such that the freeing member moves theobturating member towards the freeing position.
 15. A sampling device(2) according to claim 7, further comprising a filter (20) attached tothe container (5) so as to define, in the internal space (7), an uppercompartment into which the access opening (12) opens, and a lowercompartment, the filter (20) having pores comprised between 0.1 mm and1.5 mm.
 16. A sampling kit (1) comprising: a sampling device (2)according to claim 7, and at least one ancillary device chosen from asupply device configured to supply the internal space (7) with fluid,and a receiving device configured to receive a fluid contained in theinternal space (7), the ancillary device being in particular chosen froma reservoir of diluent fluid, an analysis tube, a distribution pipe anda collecting bag for microorganisms.
 17. A sampling kit (1) according toclaim 19, wherein the ancillary device comprises a complementaryconnection port provided with a freeing member and which is configuredto be removably connected to the connection port (26, 46) of thetransfer member (25, 45) of the sampling device (2) such that thefreeing member moves the obturating member towards the freeing position.18. A sampling device (2) according to claim 11, wherein said at leastone transfer member (25, 40) comprises at least one tube (27, 42)extending from the connection port (26, 41) and internally comprisingpart of the passage.
 19. A sampling kit (1) comprising: a samplingdevice (2) according to claim 11, and at least one ancillary devicechosen from a supply device configured to supply the internal space (7)with fluid, and a receiving device configured to receive a fluidcontained in the internal space (7), the ancillary device being inparticular chosen from a reservoir of diluent fluid, an analysis tube, adistribution pipe and a collecting bag for microorganisms.